Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray

Abstract

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Figure 1

A CART model that discriminates ER target promoters from non-targets, using a learning sample of 27 ERα targets and 97 non-ERα targets.

Figure 2

(A) AcH3 antibody (Upstate Cat. 06–942) was used to immunoprecipitate transcriptionally active chromatin components from the MDA-MB-231 (ER−) cell line. Each gene was printed three times on the array. (B) The same AcH3 antibody was used to immunoprecipitate the chromatin components from the MCF-7 (ER+) cell line. The loci that contain the active chromatin components in MCF-7, but not in the MDA-MB-231, cell are highlighted. (C) A plot of the Cy5/Cy3 ratios versus loci in both MCF7 and MDA-MB-231 cells. The ratios of MCF-7 (Cy5/Cy3)/MDA-MB-231(Cy5/Cy3) >15 suggest that the loci have active chromatin and are targets of ERα. The loci that contain the active chromatin components in MCF-7 but not MDA-MB-231 are highlighted.